- Email: [email protected]
Comments on: “DNA strand breaks” by Diem et al. [Mutat. Res. 583 (2005) 178–183] and Ivancsits et al. [Mutat. Res. 583 (2005) 184–188]
Mutation Research 603 (2006) 104–106
Letter to the Editor
Comments on: “DNA strand breaks” by Diem et al. [Mutat. Res. 583 (2005) 178–183] and Ivancsits et al. [Mutat. Res. 583 (2005) 184–188] We are writing to express our concern about the possible mis-interpretation of data in two recent publications in Mutation Research, which assessed the genotoxicity of electromagnetic fields (EMF) in a variety of human and rodent cells. In a study by Diem et al. [1], cultured human skin fibroblasts (initiated from a skin biopsy of a 6 year old healthy donor) and SV-40 transformed rat granulosa cells were exposed to 1800 MHz radiofrequency radiation (RFR) used for mobile phone communications (GSM, pulse- and talk-modulation, 2 W/kg SAR) either continuously or intermittently (5 min field on and 10 min field off) for 4, 16 and 24 h. The temperature during RFRand sham-exposures was kept at 37 ± 0.1 ◦ C. Duplicate experiments were conducted to evaluate DNA single(SSB) and double-strand breaks (DSB) using the alkaline and neutral comet assays, respectively. One thousand cells were examined at 400× magnification, manually, to categorize them into A–E, which corresponded with increased amount of DNA in the comet tail. The data were then subjected to a multiplication method to derive ‘tail factors’ which were presented in Figs. 1 and 2 (SSB), and 3 and 4 (DSB). The conclusions were: (a) both continuous and intermittent exposures induced SSB and DSB, and “the effects are much stronger at intermittent than at continuous exposure”, and (b) the induced damage is “not the result of temperature increase”. In another study from the same group, Ivancsits et al. [2] exposed lymphocytes and monocytes isolated from freshly collected human blood and cultured human skin fibroblasts, melanocytes, skeletal muscle cells as well as SV-40 transformed rat granulosa cells to extremely low frequency (ELF) EMF, 50 Hz sinusoidal, 1 mT, intermittently (5 min field on and 10 min field off) for 0–24 h. The temperature during ELF–EMF- and sham-exposures was kept at 37 ± 0.3 ◦ C. The rest of the experimental proce1383-5718/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2005.11.004
dure, data collection and analysis were similar to that in Diem et al. (1). The data were presented in Fig. 1a (SSB) and 1b (DSB). The conclusions were: (a) intermittent exposure to 50 Hz ELF–EMF has induced significantly higher levels of SSB and DSB in human fibroblasts, human melanocytes and rat granulosa cells while human lymphocytes (stimulated and unstimulated), monocytes and skeletal muscle cells did not show such response and (b) “these findings support the hypothesis that reactive oxygen species may be involved” and “this points to the possibility that differences in antioxidant capacity may be responsible for cell-specific differences in the levels of ELF–EMF-induced DNA strand breaks”. The basis of our concerns in these two reports relate to the data which were collected visually/manually, and where ‘tail factor’ was used as a surrogate marker for DNA damage. Anderson et al. [3] were the first researchers to use visual classification of comets into A–E categories, based on different levels of DNA damage within each class. This qualitative technique has been especially popular for investigators who did not have access to expensive computerized image analysis systems. However, in the reports by Diem et al. [1] and Ivancsits et al. [2], the comets in each of the A to E categories were then subjected to arbitrary transformation factors (weighted as A × 2.5, B × 12.5, C × 30.0, D × 67.5 and E × 97.5) to derive an ‘objective’ tail factor [4]. For the reasons outlined below, we express our concern that the transformation factors applied to the comet data has led to the possible mis-interpretation of the data. It is important to point out that all of the comments outlined below will also relate to the other previously published reports in which ‘tail factors’ were used to draw conclusions by these investigators [5–7]. First, continuously growing cultured cells with cell cycle durations of approximately 24–30 h (except freshly isolated cells from human blood) were used for all experiments in these studies. During prolonged RFRand ELF–EMF-exposures (especially 24 h) a number of ‘normal’ cells would have entered into the process
Letter to the Editor / Mutation Research 603 (2006) 104–106
of semi-conservative DNA synthesis (S-phase). In the alkaline comet assay, the un-winded DNA from ‘replication forks’ in S-phase cells can easily dissociate to emerge as strand breaks in the comet tail, resulting in ‘normal’ S-phase cells displaying increased comet tail length, and thus mimicking ‘damaged’ cells and would then be classified into category E. The number of S-phase cells classified into category E would have a profound impact on the ‘derived’ tail factor. Indeed, for every 1% difference in S-phase cells between sham and exposed groups, the ‘tail factor’ would change by a value of 1.0. Therefore, very small changes in the rate of cell cycle progression in exposed cells relative to the sham cells alone could account for the reported differences in ‘tail factors’. In fact, the absolute differences in tail factors between exposed and sham groups reported in Diem et al. [1] and Ivancsits et al. [2] are small. The fact that freshly isolated, non-dividing cells (human lymphocytes and monocytes) did not display changes in the tail factor [2] lends credence to the possibility that such cells may be a significant confounding variable for the derivation of the ‘tail factor’ parameter. Since the number of S-phase cells in each exposure condition was not determined in either of these two reports, the absence of the actual numbers of cells classified into groups A–E raises considerable uncertainty/doubt about the comet data and the conclusions. The second major confounder with the use of ‘tail factor’ relates to the possible inclusion of apoptotic cells in the comet data. This has serious implications for the ‘tail factor’ since apoptotic cells, which also exhibit extensive DNA fragmentation, would be visually classified into category E. Indeed, several investigators have used the comet assay to determine the incidence of apoptotic cells following exposure to genotoxic agents: an apoptotic cell will appear as a totally damaged cell with only a small amount of DNA remaining in the comet head and almost all or most of the DNA in the comet tail: see Fig. 1e in [3]; Fig. 1a in [8]; Fig. 2b in [9]; Fig. 1.12 in [10]; Fig. 1b in [11]. In the reports by Diem et al. [1] and Ivancsits et al. [2] there was no mention of the criterion used to exclude apoptotic cells in the comet data. As with the potential changes in cell cycle mentioned above, for every 1% difference in the incidence of apoptotic cells between exposed and sham groups, the tail factor would change by a value of 1.0. Our concern also relates to the statistics applied in these studies. In the study by Diem et al. [1], the data presented in Figs. 1 and 2 (SSB), 3 and 4 (DSB) show negligible standard deviations. It is not clear whether the standard deviations were calculated from a total of 2000 comets (1000 comets from each of duplicate exper-
105
iments) or from the mean of the two experiments. If the standard deviations were based on 2000 individual comet measurements, then, it is nearly assured that significant differences will be obtained between exposed and sham groups as the standard deviations generated are negligible. Indeed, it is surprising that such small standard deviations were presented in Diem et al. [1] while in the technical document describing the ‘tail factor’ transformation technique, the standard deviations reported by Diem et al. [4] were ∼25% that of the mean. Most researchers would consider the use of standard error of the means (S.E.M.) to be the appropriate variance estimator used for statistical analysis and the data from a minimum of at least three independent experiments. It must be pointed out that even if the investigators, Diem et al. [1] and Ivancsits et al. [2,5–7] had evaluated both cells in S-phase and those undergoing apoptosis, questions would still remain since the most sensitive techniques currently available to determine such cells are not accurate enough to discriminate changes, in the order of 1–2%, between exposed and sham groups. Thus, the impact of such confounding cells in category E can not be accurately determined and therefore the influence these cells have/had on subsequent ‘tail factor’ calculations adds considerable uncertainty as to the validity of ‘tail factor’ results. The increased ‘tail factors’ reported following RFR- and ELF–EMF-exposures may have resulted from changes in cell cycle, apoptosis or DNA damage or a combination thereof. Therefore, future replication and/or confirmation investigations should focus on each of these three endpoints. It is also suggested that DNA damage assessment be performed with more quantitative techniques. In conclusion, a ‘potential’ increase in the number of confounding cells (S-phase with replication forksinduced strand breaks and/or ‘apoptotic cells’ with severely fragmented DNA) in RFR- and ELF–EMFexposed cells, relative to sham-exposed samples, would certainly increase the number of cells classified into category E. For every 1% increase in confounding cells in category E the tail factor would increase by a value of 1.0. Since the numbers of these confounding cells were not determined in exposed and sham groups the validity of ‘tail factor’ data is questionable. The results and the conclusions as presented in Diem et al. [1] and Ivancsits et al. [2,5–7] will be highly ‘cited’ by researchers (in the years to come) since they are published in peer-reviewed scientific journals. However, because of the questionable nature of the results in these reports, we believe that it is imperative for researchers and public health officials wait for the data from confirmation/replication investi-
106
Letter to the Editor / Mutation Research 603 (2006) 104–106
gations to confirm or contradict these observations and to determine whether the reported changes in ‘tail factor’ are due EMF-induced DNA damage or due to other confounding variables. References [1] E. Diem, C. Schwarz, F. Adlkofer, O. Jahn, H.W. R¨udiger, Nonthermal DNA breakage by mobile-phone radiation (1800 MHz) in human fibroblasts and in transformed GFSH- R17 rat granulose cells in vitro, Mutat. Res. 583 (2005) 178– 183. [2] S. Ivancsits, A. Pilger, E. Diem, O. Jahn, H.W. R¨udiger, Cell type-specific genotoxic effects of intermittent extremely low-frequency electromagnetic fields, Mutat. Res. 583 (2005) 184–188. [3] D. Anderson, T.W. Yu, B.J. Phillips, P. Schmezer, The effect of various antioxidants and other modifying agents on oxygenradical-generated DNA damage in human lymphocytes in the COMET assay, Mutat. Res. 307 (1994) 261–271. [4] E. Diem, S. Ivancsits, H.W. R¨udiger, Basal levels of DNA strand breaks in human leukocytes determined by comet assay, J. Toxicol. Environ. Health A 65 (2002) 641–648. [5] S. Ivancsits, E. Diem, A. Pilger, H.W. R¨udiger, O. Jahn, Induction of DNA strand breaks by intermittent exposure to extremely-lowfrequency electromagnetic fields in human diploid fibroblasts, Mutat. Res. 519 (2002) 1–13. [6] S. Ivancsits, E. Diem, O. Jahn, H.W. R¨udiger, Intermittent extremely low frequency electromagnetic fields cause DNA damage in a dose dependent way, Int. Arch. Occup. Environ. Health 76 (2003) 431–436. [7] S. Ivancsits, E. Diem, O. Jahn, H.W. R¨udiger, Age-related effects on induction of DNA strand breaks by intermittent exposure to electromagnetic fields, Mech. Ageing Dev. 124 (2003) 847– 850.
[8] P.L. Olive, G. Frazer, J.P. Banath, Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay, Radiat. Res. 136 (1993) 130–136. [9] P.L. Olive, J.P. Banath, Sizing highly fragmented DNA in individual apoptotic cells using the comet assay and a DNA crosslinking agent, Exp. Cell Res. 221 (1995) 19–26. [10] N.P. Singh, Microgel electrophoresis of DNA from individual cells: principles and methodology, in: G.P. Pfeifer (Ed.), Technologies for Detection of DNA Damage and Mutations, Plenum Press, New York, 1996, pp. 3–24. [11] R.C. Wilkins, B.C. Kutzner, M. Truong, J. Sanchez-Dardon, J.R.N. McLean, Analysis of radiation-induced apoptosis in human lymphocytes: flow cytometry using annexin V and propidium iodide versus the neutral comet assay, Cytometry 48 (2002) 14–19.
Vijayalaxmi ∗ Department of Radiation Oncology, University of Texas Health Science Center, San Antonio, TX 78229, USA James P. McNamee Consumer and Clinical Radiation Protection Bureau, Health Canada, Ont., Canada Maria Rosaria Scarfi CNR-IREA, Via Diocleziano, 328-80124 Napoli, Italy ∗ Corresponding
author. E-mail address: [email protected] ( Vijayalaxmi) 19 September 2005 Available online 27 December 2005
Letter to the Editor
Comments on: “DNA strand breaks” by Diem et al. [Mutat. Res. 583 (2005) 178–183] and Ivancsits et al. [Mutat. Res. 583 (2005) 184–188] We are writing to express our concern about the possible mis-interpretation of data in two recent publications in Mutation Research, which assessed the genotoxicity of electromagnetic fields (EMF) in a variety of human and rodent cells. In a study by Diem et al. [1], cultured human skin fibroblasts (initiated from a skin biopsy of a 6 year old healthy donor) and SV-40 transformed rat granulosa cells were exposed to 1800 MHz radiofrequency radiation (RFR) used for mobile phone communications (GSM, pulse- and talk-modulation, 2 W/kg SAR) either continuously or intermittently (5 min field on and 10 min field off) for 4, 16 and 24 h. The temperature during RFRand sham-exposures was kept at 37 ± 0.1 ◦ C. Duplicate experiments were conducted to evaluate DNA single(SSB) and double-strand breaks (DSB) using the alkaline and neutral comet assays, respectively. One thousand cells were examined at 400× magnification, manually, to categorize them into A–E, which corresponded with increased amount of DNA in the comet tail. The data were then subjected to a multiplication method to derive ‘tail factors’ which were presented in Figs. 1 and 2 (SSB), and 3 and 4 (DSB). The conclusions were: (a) both continuous and intermittent exposures induced SSB and DSB, and “the effects are much stronger at intermittent than at continuous exposure”, and (b) the induced damage is “not the result of temperature increase”. In another study from the same group, Ivancsits et al. [2] exposed lymphocytes and monocytes isolated from freshly collected human blood and cultured human skin fibroblasts, melanocytes, skeletal muscle cells as well as SV-40 transformed rat granulosa cells to extremely low frequency (ELF) EMF, 50 Hz sinusoidal, 1 mT, intermittently (5 min field on and 10 min field off) for 0–24 h. The temperature during ELF–EMF- and sham-exposures was kept at 37 ± 0.3 ◦ C. The rest of the experimental proce1383-5718/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2005.11.004
dure, data collection and analysis were similar to that in Diem et al. (1). The data were presented in Fig. 1a (SSB) and 1b (DSB). The conclusions were: (a) intermittent exposure to 50 Hz ELF–EMF has induced significantly higher levels of SSB and DSB in human fibroblasts, human melanocytes and rat granulosa cells while human lymphocytes (stimulated and unstimulated), monocytes and skeletal muscle cells did not show such response and (b) “these findings support the hypothesis that reactive oxygen species may be involved” and “this points to the possibility that differences in antioxidant capacity may be responsible for cell-specific differences in the levels of ELF–EMF-induced DNA strand breaks”. The basis of our concerns in these two reports relate to the data which were collected visually/manually, and where ‘tail factor’ was used as a surrogate marker for DNA damage. Anderson et al. [3] were the first researchers to use visual classification of comets into A–E categories, based on different levels of DNA damage within each class. This qualitative technique has been especially popular for investigators who did not have access to expensive computerized image analysis systems. However, in the reports by Diem et al. [1] and Ivancsits et al. [2], the comets in each of the A to E categories were then subjected to arbitrary transformation factors (weighted as A × 2.5, B × 12.5, C × 30.0, D × 67.5 and E × 97.5) to derive an ‘objective’ tail factor [4]. For the reasons outlined below, we express our concern that the transformation factors applied to the comet data has led to the possible mis-interpretation of the data. It is important to point out that all of the comments outlined below will also relate to the other previously published reports in which ‘tail factors’ were used to draw conclusions by these investigators [5–7]. First, continuously growing cultured cells with cell cycle durations of approximately 24–30 h (except freshly isolated cells from human blood) were used for all experiments in these studies. During prolonged RFRand ELF–EMF-exposures (especially 24 h) a number of ‘normal’ cells would have entered into the process
Letter to the Editor / Mutation Research 603 (2006) 104–106
of semi-conservative DNA synthesis (S-phase). In the alkaline comet assay, the un-winded DNA from ‘replication forks’ in S-phase cells can easily dissociate to emerge as strand breaks in the comet tail, resulting in ‘normal’ S-phase cells displaying increased comet tail length, and thus mimicking ‘damaged’ cells and would then be classified into category E. The number of S-phase cells classified into category E would have a profound impact on the ‘derived’ tail factor. Indeed, for every 1% difference in S-phase cells between sham and exposed groups, the ‘tail factor’ would change by a value of 1.0. Therefore, very small changes in the rate of cell cycle progression in exposed cells relative to the sham cells alone could account for the reported differences in ‘tail factors’. In fact, the absolute differences in tail factors between exposed and sham groups reported in Diem et al. [1] and Ivancsits et al. [2] are small. The fact that freshly isolated, non-dividing cells (human lymphocytes and monocytes) did not display changes in the tail factor [2] lends credence to the possibility that such cells may be a significant confounding variable for the derivation of the ‘tail factor’ parameter. Since the number of S-phase cells in each exposure condition was not determined in either of these two reports, the absence of the actual numbers of cells classified into groups A–E raises considerable uncertainty/doubt about the comet data and the conclusions. The second major confounder with the use of ‘tail factor’ relates to the possible inclusion of apoptotic cells in the comet data. This has serious implications for the ‘tail factor’ since apoptotic cells, which also exhibit extensive DNA fragmentation, would be visually classified into category E. Indeed, several investigators have used the comet assay to determine the incidence of apoptotic cells following exposure to genotoxic agents: an apoptotic cell will appear as a totally damaged cell with only a small amount of DNA remaining in the comet head and almost all or most of the DNA in the comet tail: see Fig. 1e in [3]; Fig. 1a in [8]; Fig. 2b in [9]; Fig. 1.12 in [10]; Fig. 1b in [11]. In the reports by Diem et al. [1] and Ivancsits et al. [2] there was no mention of the criterion used to exclude apoptotic cells in the comet data. As with the potential changes in cell cycle mentioned above, for every 1% difference in the incidence of apoptotic cells between exposed and sham groups, the tail factor would change by a value of 1.0. Our concern also relates to the statistics applied in these studies. In the study by Diem et al. [1], the data presented in Figs. 1 and 2 (SSB), 3 and 4 (DSB) show negligible standard deviations. It is not clear whether the standard deviations were calculated from a total of 2000 comets (1000 comets from each of duplicate exper-
105
iments) or from the mean of the two experiments. If the standard deviations were based on 2000 individual comet measurements, then, it is nearly assured that significant differences will be obtained between exposed and sham groups as the standard deviations generated are negligible. Indeed, it is surprising that such small standard deviations were presented in Diem et al. [1] while in the technical document describing the ‘tail factor’ transformation technique, the standard deviations reported by Diem et al. [4] were ∼25% that of the mean. Most researchers would consider the use of standard error of the means (S.E.M.) to be the appropriate variance estimator used for statistical analysis and the data from a minimum of at least three independent experiments. It must be pointed out that even if the investigators, Diem et al. [1] and Ivancsits et al. [2,5–7] had evaluated both cells in S-phase and those undergoing apoptosis, questions would still remain since the most sensitive techniques currently available to determine such cells are not accurate enough to discriminate changes, in the order of 1–2%, between exposed and sham groups. Thus, the impact of such confounding cells in category E can not be accurately determined and therefore the influence these cells have/had on subsequent ‘tail factor’ calculations adds considerable uncertainty as to the validity of ‘tail factor’ results. The increased ‘tail factors’ reported following RFR- and ELF–EMF-exposures may have resulted from changes in cell cycle, apoptosis or DNA damage or a combination thereof. Therefore, future replication and/or confirmation investigations should focus on each of these three endpoints. It is also suggested that DNA damage assessment be performed with more quantitative techniques. In conclusion, a ‘potential’ increase in the number of confounding cells (S-phase with replication forksinduced strand breaks and/or ‘apoptotic cells’ with severely fragmented DNA) in RFR- and ELF–EMFexposed cells, relative to sham-exposed samples, would certainly increase the number of cells classified into category E. For every 1% increase in confounding cells in category E the tail factor would increase by a value of 1.0. Since the numbers of these confounding cells were not determined in exposed and sham groups the validity of ‘tail factor’ data is questionable. The results and the conclusions as presented in Diem et al. [1] and Ivancsits et al. [2,5–7] will be highly ‘cited’ by researchers (in the years to come) since they are published in peer-reviewed scientific journals. However, because of the questionable nature of the results in these reports, we believe that it is imperative for researchers and public health officials wait for the data from confirmation/replication investi-
106
Letter to the Editor / Mutation Research 603 (2006) 104–106
gations to confirm or contradict these observations and to determine whether the reported changes in ‘tail factor’ are due EMF-induced DNA damage or due to other confounding variables. References [1] E. Diem, C. Schwarz, F. Adlkofer, O. Jahn, H.W. R¨udiger, Nonthermal DNA breakage by mobile-phone radiation (1800 MHz) in human fibroblasts and in transformed GFSH- R17 rat granulose cells in vitro, Mutat. Res. 583 (2005) 178– 183. [2] S. Ivancsits, A. Pilger, E. Diem, O. Jahn, H.W. R¨udiger, Cell type-specific genotoxic effects of intermittent extremely low-frequency electromagnetic fields, Mutat. Res. 583 (2005) 184–188. [3] D. Anderson, T.W. Yu, B.J. Phillips, P. Schmezer, The effect of various antioxidants and other modifying agents on oxygenradical-generated DNA damage in human lymphocytes in the COMET assay, Mutat. Res. 307 (1994) 261–271. [4] E. Diem, S. Ivancsits, H.W. R¨udiger, Basal levels of DNA strand breaks in human leukocytes determined by comet assay, J. Toxicol. Environ. Health A 65 (2002) 641–648. [5] S. Ivancsits, E. Diem, A. Pilger, H.W. R¨udiger, O. Jahn, Induction of DNA strand breaks by intermittent exposure to extremely-lowfrequency electromagnetic fields in human diploid fibroblasts, Mutat. Res. 519 (2002) 1–13. [6] S. Ivancsits, E. Diem, O. Jahn, H.W. R¨udiger, Intermittent extremely low frequency electromagnetic fields cause DNA damage in a dose dependent way, Int. Arch. Occup. Environ. Health 76 (2003) 431–436. [7] S. Ivancsits, E. Diem, O. Jahn, H.W. R¨udiger, Age-related effects on induction of DNA strand breaks by intermittent exposure to electromagnetic fields, Mech. Ageing Dev. 124 (2003) 847– 850.
[8] P.L. Olive, G. Frazer, J.P. Banath, Radiation-induced apoptosis measured in TK6 human B lymphoblast cells using the comet assay, Radiat. Res. 136 (1993) 130–136. [9] P.L. Olive, J.P. Banath, Sizing highly fragmented DNA in individual apoptotic cells using the comet assay and a DNA crosslinking agent, Exp. Cell Res. 221 (1995) 19–26. [10] N.P. Singh, Microgel electrophoresis of DNA from individual cells: principles and methodology, in: G.P. Pfeifer (Ed.), Technologies for Detection of DNA Damage and Mutations, Plenum Press, New York, 1996, pp. 3–24. [11] R.C. Wilkins, B.C. Kutzner, M. Truong, J. Sanchez-Dardon, J.R.N. McLean, Analysis of radiation-induced apoptosis in human lymphocytes: flow cytometry using annexin V and propidium iodide versus the neutral comet assay, Cytometry 48 (2002) 14–19.
Vijayalaxmi ∗ Department of Radiation Oncology, University of Texas Health Science Center, San Antonio, TX 78229, USA James P. McNamee Consumer and Clinical Radiation Protection Bureau, Health Canada, Ont., Canada Maria Rosaria Scarfi CNR-IREA, Via Diocleziano, 328-80124 Napoli, Italy ∗ Corresponding
author. E-mail address: [email protected] ( Vijayalaxmi) 19 September 2005 Available online 27 December 2005